The blood red color, verging on black at the base, displayed by the petals of flowers of Adonis aestivalis and Adonis annua results from the accumulation of carotenoid pigments (Egger, 1965; Neamtu et al., 1966; Seybold and Goodwin, 1959), predominantly the ketocarotenoid astaxanthin (3,3′-dihydroxy-4,4′-diketo-β,β-carotene; FIG. 1). The biosynthesis of astaxanthin occurs in a number of bacteria and fungi (Goodwin, 1980; Johnson and An, 1991), and in certain unicellular algae (Goodwin, 1980; Grung and Liaaen-Jensen, 1993; Johnson and An, 1991; Orosa et al., 2000). Astaxanthin has been found in a few other plant species (Czeczuga, 1987; Goodwin, 1980), but no other plant produces this ketocarotenoid in as great a quantity as in Adonis flowers [ca. 1% of dry weight for the flower petals of Adonis annua according to Renstrøm et al., (1981)].
Astaxanthin has found use as a topical antioxidant (in sun blocking lotions, for example) and as an ingredient of human nutritional supplements. See U.S. Pat. No. 6,433,025 to Lorenz. This carotenoid, however, is perhaps best known for providing an attractive orange-red color to the flesh of wild salmon and other fish (Shahidi et al, 1998) and a blue hue (changing to red upon boiling as the proteins that bind astaxanthin are denatured) to the carapace of lobster and of other crustaceans (Chayen et al., 2003; Tanaka et al., 1976).
Fish and crustaceans that are raised in captivity require the addition of astaxanthin to their feed in order to acquire the appropriate coloration. The substantial and expanding market for astaxanthin as a fish feed additive is supplied largely by chemical synthesis, but there is considerable interest in the development of a biological production process to provide an alternative source of this valuable ketocarotenoid. The green alga Haematococcus pluvialis (Lorenz and Cysewski, 2000; Orosa et al., 2000) and the fungus Xanthophyllomyces dendrorhous (formerly known as Phaffia rhodozyma; Johnson, 2003; Visser et al., 2003,) have received the most attention in this regard. See also U.S. Pat. No. 6,413,736 to Jacobson et al., and incorporated by reference herein as if set forth in its entirety. However, the cost of producing astaxanthin in these organisms remains much greater than that for astaxanthin produced by chemical synthesis.
Currently, synthetic astaxanthin is added to feeds prepared for production of salmonids and red sea bream in aquaculture to provide a source of this carotenoid compound. See, for example, U.S. Pat. No. 5,739,006 to Abe et al. In some cases, synthetic canthaxanthin (an oxygenated carotenoid compound that is very closely related to astaxanthin) is used in place of astaxanthin in feeds for salmonids and red sea bream, but this compound does not add the appropriate color to these fishes as efficiently as the naturally predominant astaxanthin.
Recently, attempts have been made, with limited success, to engineer plants for astaxanthin production by introduction of genes from algal and/or bacterial carotenoid pathways (Mann et al., 2000; Ralley et al., 2004; Stålberg et al., 2003). Problems encountered with this strategy include: an incomplete conversion of precursors (i.e. β-carotene and zeaxanthin) into astaxanthin, competition of the introduced bacterial or green algal enzymes with endogenous enzymes that also use β-carotene and/or zeaxanthin as substrates (i.e. zeaxanthin epoxidase), and the accumulation of unwanted intermediates of the pathway (i.e. adonixanthin and adonirubin).
A few attempts have been made to develop and exploit Adonis aestivalis as a source of astaxanthin for the pigmentation of fish (Kamata et al., 1990; Rodney, 1995), and this plant is currently grown in China expressly for this purpose. However, despite high concentrations of astaxanthin in the flower petals, a relatively low yield of petal biomass per acre makes Adonis a less than ideal vehicle for biological production of this pigment. An understanding of the biosynthetic pathway leading to astaxanthin in Adonis aestivalis would enable the pathway to be transferred to other plants, such as marigold, that could provide a much greater yield of carotenoid-containing biomass and, therefore, a much less costly source of natural astaxanthin.
From zeaxanthin (3,3′-dihydroxy-β,β-carotene), a dihydroxy carotenoid present in the green tissues of most higher plants, the formation of astaxanthin requires only that a carbonyl be introduced at the number 4 carbon of each ring (FIG. 1). As a practical matter, the addition of the carbonyl may need to occur prior to hydroxylation of the ring [i.e. β-carotene rather than zeaxanthin would be the substrate for the enzyme, and echinenone (4-keto-β,β-carotene) and canthaxanthin (4,4′-diketo-β,β-carotene) would be the immediate products (Breitenbach et al., 1996; Fraser et al., 1998; Lotan and Hirschberg, 1995)]. Enzymes that catalyze carbonyl addition at the number 4 carbon of carotenoid β-rings have so far been identified in bacteria (De Souza et al., 2002; Harker and Hirschberg, 1999; Misawa et al., 1995a and 1995b), photosynthetic bacteria (Hannibal et al., 2000), cyanobacteria (Fernandez-Gonzalez et at, 1997; Steiger and Sandmann, 2004), and green algae (Kajiwara et al., 1995; Lotan and Hirschberg, 1995). The green algal enzymes that have been characterized are orthologs of those found in bacteria, in photosynthetic bacteria, and in certain of the cyanobacteria, as evidenced by the significant similarity of their amino acid sequences. The “4-ketolase” enzyme of the cyanobacterium Synechocystis sp. PCC6803 is distinctly different from these others (Fernandez-Gonzalez et al., 1997). It is related instead to an enzyme that catalyzes an earlier step in the carotenoid pathway of Synechocystis: the carotene isomerase (Breitenbach et al., 2001; Masamoto et al., 2001). What appears to be a third type of 4-ketolase enzyme, found in the fungus Xanthophyllomyces dendrorhous (Phaffia rhodozyma), is related to cytochrome P450 enzymes (Hoshino et al., 2002). The activity of this enzyme has not yet been demonstrated directly. The enzyme's putative function as an “astaxanthin synthase” has been attributed on the basis of genetic complementation experiments. The gene encoding this enzyme restores the ability to synthesize astaxanthin in a X. dendrorhous mutant that accumulates only β-carotene (Hoshino et al., 2002). Because no mutants have been found that accumulate any of the intermediates between β-carotene and astaxanthin (Visser et al., 2003), it is thought that the product of this gene is responsible for both 3-hydroxylation and 4-keto addition.
The green plant Adonis aestivalis synthesizes carotenoids with 4-keto-β-rings via a biochemical pathway unrelated to any yet characterized or described. The present inventor has previously disclosed (U.S. Pat. No. 6,551,807 to Cunningham) two nucleic acid sequences from Adonis aestivalis (FIG. 2 and FIG. 3; SEQ ID NO: 1 and SEQ ID NO: 2) that encode enzymes (FIG. 4; SEQ ID NO: 3 and SEQ ID NO: 4) which convert β-carotene into carotenoids with ketocarotenoid-like absorption spectra (i.e. red-shifted and with a diminution of spectral fine structure). More recent work (Cunningham and Gantt, 2005) has demonstrated that the Adonis aestivalis “ketolase” enzymes described in this earlier patent (AdKeto1 and AdKeto2) each catalyze two different reactions: a desaturation of carotenoid β-rings at the 3-4 position and a hydroxylation at the number 4 carbon. The inventor now discloses herein the DNA sequence of an Adonis aestivalis cDNA that encodes an enzyme, referred to as AdKC28, that works in concert with either one of the two 3,4-desaturase/4-hydroxylase enzymes previously described (AdKeto1 and AdKeto2) to convert β-carotene into astaxanthin.